CN106755088A - 一种自体car‑t细胞制备方法及应用 - Google Patents
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Abstract
本发明涉及一种自体CAR‑T细胞制备方法及其应用。是将构建的CD28‑CD137‑CD19‑CD3全长基因,用CRISPR/Cas9技术导入到患者T细胞制作成CAR‑T细胞,然后经过体外扩增后,回输到患者体内进行抗肿瘤治疗。与传统肿瘤治疗方法相比,该方法为细胞靶向性治疗,副作用小,而且经过基因修饰的T细胞可在其表面稳定的表达抗原结合域,识别靶抗原的同时,无MHC限制性,提高了肿瘤的治疗效果。
Description
技术领域
本发明涉及医学、免疫学、细胞生物学和分子生物学领域,具体涉及一种自体CAR-T细胞制备方法及其应用。
背景技术
免疫细胞疗法作为一种新的癌症治疗方法,其治疗效果在临床上得到越来越多的证实,CAR-T细胞(嵌合抗原受体T细胞)技术是利用病人自身的免疫细胞来清除癌细胞免疫细胞疗法,在多种血液癌症治疗中展现了极高的优势,攻破实体瘤也指日可待,由于肿瘤免疫疗法在治愈肿瘤的同时又没有传统放疗、化疗的治疗毒性,所以免疫细胞疗法在肿瘤的治疗中具有广阔的前景。
过继细胞治疗(ACT)因其在短时间内扩增和活化具有抗瘤活性的效应细胞,在临床研究中备受关注。TIL、CIK、NK、NKT和γδT这些用于ACT的细胞在临床应用研究中取得一定的疗效,但肿瘤抗原特异性、亲和力好的免疫细胞来源困难、数量较少,杀瘤活性和体内持续时间不足,制约了ACT的发展。
T细胞可携带外源基因安全的进入人体内(Kerkar SP."Model T"cells:a time-tested vehicle for gene therapy Fronti Immunol,2012;4:304-304)。T细胞容易获得,在体外可被有效地诱导,并能大量增殖,可根据T细胞的这些特点,采用现代基因转导技术,赋予T细胞新的特性,经过修饰的T细胞可在其表面稳定的表达抗原结合域,识别靶抗原的同时,无MHC限制性(Ito F,Chang AE(2013).Cancer immunotherapy:current status andfuture Directions.Surg Onco Clin N Am,22(4):765-783.)。
于是,研究者尝试使用基因修饰T细胞来解决制约ACT发展的问题,用来修饰T细胞的基因有TCR、CAR、促进免疫细胞增殖的细胞因子(如IL-2、IL-15)等。其中嵌合抗原受体T细胞(CAR-T)是以能编码单链抗体-共刺激分子-免疫受体酪氨酸活化基因序列的嵌合分子的融合基因修饰T细胞。因其具有肿瘤抗原识别特异性强、亲和力高、非MHC限制性及可在体内外大量扩增的优点而受到较多关注。
最新研究表明,采用初始T细胞(TN)或者中心记忆T细胞(TCM),这些细胞比分化的T细胞具有更强大的体内杀伤活性。具体操作:可通过类T细胞干细胞标记物CD62L进行T细胞分选。例如,将来自癌症患者的PBMC与自体肿瘤细胞和磁珠共同培养,所述磁珠能通过CD3与CD28、CD40或CD28加CD40的组合来活化T细胞。
CAR是人工构建的融合基因编码的跨膜分子,有胞外区、胞内区和跨膜区构成。胞外区的单链可变区(scFv),负责特异性抗原的识别;胞内区负责信号的转导,当胞外区和抗原特异性结合后,胞内区启动细胞活化所需的信号,从而促进T细胞增殖、释放细胞因子、抗细胞凋亡等;跨膜区链接胞外区和胞内区,对每一个区域的不同设计直接影响CAR-T细胞功能的发挥。
B淋巴细胞白血病和恶性淋巴瘤是原发于骨髓造血系统和淋巴结并弥漫全身的恶性肿瘤。传统的放化疗虽然有一定疗效,但没有选择性,对正常组织损伤很大。近年来,生物治疗方法广泛应用于肿瘤治疗,尤其是单克隆抗体,如rituximab并因其特异靶向性、高亲和性而收到了良好效果。单抗通过Fc段与效应细胞表面激活性受体FcγRⅠ/FcγRⅢ结合,从而介导杀伤作用,但具有免疫杀伤作用的T细胞因为表面缺乏上述受体而不能被有效介导,从而削弱了机体对肿瘤的免疫效应。用抗CD19修饰的CAR-T细胞能同时识别多种不同抗原,可以介导T细胞高效特异杀伤B淋巴细胞来源的肿瘤细胞,为解决肿瘤的免疫逃逸带来了曙光。
CD19是较为理想的肿瘤相关抗原,它表达于除干细胞以外的B淋巴细胞发育的各个阶段,因此B细胞来源的恶性细胞均有CD19表达。有关研究表明CD19抗体scFv构建的CAR-T细胞具有高度亲和力和裂解原代B细胞急性淋巴细胞白血病(B-ALL)瘤细胞的作用(Cooper LJ,Topp MS,Serrano LM,et al.T-cell clones can be rendered specificfor CD19:toward the selective augmentation of the graft-versus-B-lineageleukemia effect.Blood,2003;101(4):1637-1644.)。Kalos等人在用抗CD19修饰的第二代CAR-T细胞进行治疗进展性CLL时发现,CAR-T细胞在体内扩增速度快甚至可达1000倍以上,持续时间超过6个月。不但消除了肿瘤细胞,一部分细胞还以记忆性CAR-T在体内持续存在(Kalos M,Levine BL,Porter DL,et al.T cells with chimeric antigen receptorshave potent antitumor effects and can establish memory in patients withadvanced leukemia.Sci Transl Med,2011;3(95):95ra73-95ra73)。B细胞成熟抗原表达于成熟B细胞和浆细胞表面,能促进两者的存活,B细胞成熟抗原是肿瘤坏死因子超家族成员,可结合B细胞活化因子和增殖诱导配体,在治疗多发性骨髓瘤上是一个有前景的靶目标。
CD3是T淋巴细胞表面特异分子,功能是把TCR与外来结合的抗原信息传递到细胞内,启动细胞内的活化过程,在T细胞接受抗原刺激后发生激活的早期过程起重要作用。
4-1BB又称CD137,是T细胞表面活化分子,在活化的T细胞上表达,4-1BB的参与可以放大已经被诱导的免疫应答。4-1BB与抗4-1BB的单克隆抗体相接触后可以刺激被抗原激活的、有肿瘤特异性杀伤活性的CD8+T淋巴细胞增殖,刺激干扰素-γ(IFN-γ)和其他Th1型细胞因子(IL-2),肿瘤坏死因子(TNF-α)的生成、释放,并刺激对抗细胞凋亡的T细胞的保护。另外4-1BB有免疫调节效应。4-1BB配体(4-1BBL)可显著放大CD8+T淋巴细胞的应答。研究显示,在CAR中间加入共刺激分子4-1BB后得到的CAR-T细胞具有分泌跟过细胞因子和更强的增殖活性(Carpenito C,Milone MC,Hassan R,et al.Control of large,established tumor xenografts with genetically retargeted human T cellscontaining CD28and CD137domains.Proc Natl Acad Sci USA,2009;106(9):3360-3365)。有关研究表明,CD28的跨膜区表达CAR的能力最强。CD28+4/1BB组合提供共刺激信号,当抗原刺激第一信号通路后,可以上调抗细胞凋亡基因的表达、促进IL2分泌。
有关研究表明,CAR引入共刺激分子信号(如CD28、CD137、CD27、CD244等),可提高T细胞的细胞毒性、增殖活性、存活时间,增加抗原诱导的细胞因子释放,上调抗细胞凋亡蛋白。这些作用可增强T细胞对目标肿瘤的杀伤作用(Song DG,Ye Q,Poussin M,etal.CD27costimulation augments the survival and antitumor activity ofredirected human T cells in vivo.Blood,2012;119(3):696-706)
因此可通过基因修饰,定向构建scFv–CD28-CD137-CD19-CD3基因,然后将其导入到T细胞制作成CAR-T细胞,从而可以实现介导T细胞特异性杀伤B淋巴细胞来源的肿瘤细胞,而且共刺激分子信号还可以提高CAR-T细胞的细胞毒性、增殖活性,增强T细胞对目标肿瘤细胞的杀伤作用。
CAR-T细胞的制备过程中,需要将抗体序列及T细胞受体信号途径的编码分子相关的序列导入至T细胞中,外源基因导入进行治疗,大多数研究结果显示,这是一种比较安全的方式。目前美国用于临床试验导入外源基因的方式大致有逆转录病毒、慢病毒、转座子mRNA、电转入等方式。早在1990年,Rosenberg小组提出利用逆转录病毒将外源基因导入T细胞进行肿瘤是安全可行的,同年美国FDA批准了基因治疗。逆转录病毒感染的主要是分裂期细胞,对于非分裂细胞感染能力极弱。在此基础上发展出的慢病毒,则对分裂和非分裂细胞均具有较好的感染能力。采用传统直接转染的方式难以将外源基因导入至T淋巴细胞中,因此绝大部分CAR-T细胞制备时,采用的慢病毒系统,因为病毒类载体涉及较多专利上的争议,因此研究者们又尝试采用转座子结合电转导入外源基因来制备CAR-T细胞,临床前研究也证实该方法是可行的,尽管逆转录病毒和慢病毒导入外源基因进行治疗被认为是安全的,但是由于外源基因却是随机整合至宿主基因组并在细胞内长期存在且稳定表达,存在不合适的插入位点突变,可能会引起细胞发生转化的潜在风险,转座子电转的方式也存在着类似的风险。因此,有研究探索将编码质粒直接通过电转导入细胞内,但是因为质粒导入的方式大多数为瞬时表达,能否达到治疗效果还有待进一步实验验证。利用RNA作为目标导向的CRISPR/Cas9基因编辑技术被认为是基因编辑技术最新的成果,研究发现Cas9可以解旋DNA,解旋之后gRNA和靶向DNA配对,并利用配对结合所释放的能量进行切割DNA。
表达CAR的T细胞可以直接识别并结合肿瘤细胞表面的TAA,CAR将信号传入T细胞内,激活T细胞分泌细胞因子包括穿孔素、颗粒酶、INF-γ、TNF-α等,从而发挥杀伤肿瘤细胞作用。因此,CAR-T细胞是MHC非限制性的。CAR-T细胞将抗体-抗原特异性结合能力以及细胞介导的杀伤功能结合于一体,制作简单,应用广泛,是免疫抗肿瘤治疗的重要方法。
CAR将抗体对肿瘤抗原的高亲和性和T细胞的杀伤机制结合,通过基因转染T淋巴细胞,使其能特异性的杀伤肿瘤细胞。
发明内容
本发明的目的是在肿瘤患者体外生产转基因T淋巴细胞,然后扩增后输入患者体内用于治疗肿瘤疾病。具体做法是用慢病毒做载体将构建的CD28-CD137-CD19-CD3全长基因,转染T细胞制作成CAR-T细胞,然后经过体外扩增后,回输到患者体内进行肿瘤治疗。
一种自体CAR-T细胞制备方法,包括步骤如下:
(a)从肿瘤患者体内获得细胞样本并分离和激活,所述样本为T细胞或T细胞的祖细胞;
(b)构建scFv–CD28-CD137-CD19-CD3;
(c)制作pCAG-T7-CD28-CD137-CD19-CD3-gRNA重组质粒
(d)转染T细胞;
(e)体外培养扩增CAR-T细胞群体;
(f)CAR-T细胞回输患者体内,进行抗肿瘤治疗;
所述步骤(a)的具体操作流程为:1)抗凝血管抽取骨髓瘤患者外周血;2)向管中加入红细胞裂解液和等体积PBS,轻轻吹打成细胞悬液;3)另取两支离心管,加入LTS1077淋巴细胞分层液;用吸管吸取细胞悬液,在距离淋巴细胞分层液上方0.5-3cm处将细胞悬液小心而缓慢的加入,使细胞悬液重叠与淋巴细胞分层液上,2000r/min离心5-50min;4)取出离心管,移液管吸去最上层的血浆,移液枪吸取血装层下的单个核细胞置入离心管中,加入PBS,轻轻吹打均匀后再次离心,1500r/min,10min,去掉上清液,共洗涤2-5次;5)向去掉上清后的离心管内加入含灭活胎牛血清、青链霉素和RPMI-1640的培养基放置于37℃细胞培养箱中扩大培养;6)离心,收取外周血单核细胞,置于液氮罐中保存备用。
所述细胞裂解液和PBS等体积,细胞裂解液和PBS的总体积与抽取的外周血体积比为2:1-1:2。
淋巴细胞分层液和细胞悬液的体积比为2:1-1:2;步骤4)中加入PBS的体积为淋巴细胞分层液的1/2-3倍;
步骤5)中加入的浓度分别是10%灭活胎牛血清、100U/ml青链霉素、100U/mlIL-2的RPMI-1640。
所述步骤(c)为根据CRISPR/Cas9靶点设计原则,应用crispr在线工具设计、合成靶向scFv–CD28-CD137-CD19-CD3全长基因的gRNA和引物,并将其插入到CRISPR/Cas9质粒骨架载体中,制作成pCAG-T7-CD28-CD137-CD19-CD3-gRNA重组质粒。
所述步骤(d)为将步骤(c)制得的质粒CAG-T7-CD28-CD137-CD19-CD3-gRNA与步骤(a)制得的外周血单核细胞混合进行转染。
所述步骤(e)的详细步骤为:用OKM-100细胞培养液调整接种密度为(1-2)×106/ml后加入到含有刺激因子细胞培养瓶内,然后再加入自体灭活血浆,至细胞培养箱内培养,培养4~5d待细胞铺满瓶底后,将细胞转入大的细胞培养瓶中继续培养,培养2~3d后,将大的细胞培养瓶中的细胞转至含有更大的OKM-200细胞培养液的CO2透气培养袋中继续培养,培养6-7d后,收获细胞。
本发明的优点在于:
1、与传统肿瘤治疗方法相比,该方法副作用小。
2、经过基因修饰的T细胞可在其表面稳定的表达抗原结合域,识别靶抗原的同时,无MHC限制性。
3、CAR-T细胞在体内扩增速度快,持续时间长。不但可消除肿瘤细胞,一部分细胞还以记忆性CAR-T在体内持续存在。
4、CAR引入共刺激分子信号(如CD28、CD137、CD3、CD19等),可提高T细胞的细胞毒性、增殖活性、存活时间,增加抗原诱导的细胞因子释放,上调抗细胞凋亡蛋白,这些作用可增强T细胞对目标肿瘤的杀伤作用。
5本发明用CD19抗体scFv基因构建的CAR-T细胞具有高度亲和力和裂解原代B细胞急性淋巴细胞白血病(B-ALL)瘤细胞的作用,B细胞成熟抗原是肿瘤坏死因子超家族成员,可结合B细胞活化因子和增殖诱导配体,在治疗多发性骨髓瘤上是一个有前景的靶目标。
6、CAR-T细胞体外扩增培养方法操作简单,培养时间较短,(2-3周)。
7、以癌症患者自体淋巴细胞进行构建CAR-T细胞,不存在违反医学伦理的风险。
8、使用CRISPR/Cas9基因编辑技术插入目标基因,操作便捷,准确率高。
具体实施方式
为了更详细地说明本发明,给出下述制备实例。但本发明的范围并不局限于此。
实施例1
本发明提供了一种自体CAR-T淋巴细胞的制备方法,具体方案包括:
(a)从肿瘤患者获得细胞样本,所述样本包括T细胞或T细胞的祖细胞;
(b)构建scFv–CD28-CD137-CD19-CD3,
(c)制作pCAG-T7-CD28-CD137-CD19-CD3-gRNA重组质粒
(d)转染T细胞
(e)体外培养扩增CAR-T细胞群体;
(f)CAR-T细胞回输患者体内,进行抗肿瘤治疗
(a)、外周血单核细胞的分离与激活
选取肝肾功能正常;PBMC对CD3/CD28刺激有良好应答的骨髓瘤癌症患者。
1、抗凝血管抽取骨髓瘤患者外周血10ml;
2、向管中加入红细胞裂解液和等体积PBS,轻轻吹打成细胞悬液20ml;
3、另取两支50ml离心管,加入10mlLTS1077淋巴细胞分层液。用吸管吸取10ml细胞悬液,在距离淋巴细胞分层液上方1cm处将细胞悬液小心而缓慢的加入,使细胞悬液重叠与淋巴细胞分层液上,2000r/min离心20min;
4、取出离心管,移液管吸去最上层的血浆,移液枪吸取血装层下的单个核细胞置入离心管中,加入10mlPBS,轻轻吹打均匀后再次离心,1500r/min,10min,去掉上清液,共洗涤3次。
5、向去掉上清后的离心管内加入含10%灭活胎牛血清、100U/ml青链霉素、100U/mlIL-2的RPMI-1640培养基放置于37℃,5%CO2的细胞培养箱中扩大培养。
6、离心,收取外周血单核细胞,置于液氮罐中保存备用。
(b)Hinge-TM-CD28-CD137-CD19-CD3ξ的设计
CAR的较链区(Hinge)和跨膜区(TM)来自CD8a(aa135-205,GenBank:BC025715.1)、CD28功能区(aa180-220,GenBank:BC025715.1)、CD137(aa214-255,GenBank:U03397.1)、CD19(aa422-433,深圳欣博盛生物科技)及CD3ξ(aa52-163,Genbank:J04132.1),Hinge-TM-CD28-CD137-CD19-CD3ξ表达框通过基因合成仪(Dr.Oligo192合成仪)完成。
Hinge-TM-CD28-CD137-CD19-CD3ξ扩增
Hinge-TM-CD28-CD137-CD19-CD3ξ扩增引物:
F2:5-TGGCACCAAGCTGGAAATCAAAACCACGACGCCAGCGCCGCGA-3
R2:5-CGGGATCCTTAGCGAGGGGGCAGGGCCT-3,引物由上海生工生物有限公司合成。
获得的目的基因片段Hinge-TM-CD28-CD137-CD19-CD3ξ,分装冻存于-20℃冰箱中保存。
(c)制作pCAG-T7-CD28-CD137-CD19-CD3-gRNA重组质粒
根据CRISPR/Cas9靶点设计原则,设计出符合要求的gRNA,对其进行筛选及脱靶效应评估,挑选出特性强的gRNA。然后利用生物信息软件进行全基因组比对,避免选择脱靶风险大的靶点序列,进行体外以增加成功率。
将上述步骤获得的Hinge-TM-CD28-CD137-CD19-CD3ξ与载体质粒进行连接,用连接酶将大小为2436bp的目的片段Hinge-TM-CD28-CD137-CD19-CD3ξ连接入载体基因中。反应体系为Solution I酶连接体系
Solution I酶连接体系
PCR反应条件:95℃反应3分钟,95℃缓慢自然冷却至25℃,16℃反应5分钟,然后将退货产物连接到载体中:pCAG-T7 1μl,PCR反应产物2μl,用水稀释至10μl,充分混合后,室温(25℃)静置5分钟。取连接产物5μl加入到解冻的50μl DH5α感受态细胞中,轻弹混匀,冰浴30分钟后,42℃热激90秒,冰上静置2分钟,直接涂于平板,第二天,挑出生长良好的菌落于LB培养液中,37℃200rpm摇床培养过夜。取5ml菌液进行测序。
2、pCAG-T7-CD28-CD137-CD19-CD3-gRNA对T细胞的转染
将构建好的重组质粒用TE buffer稀释至等浓度后等体积混合,将步骤(a)获得的生长良好的单核细胞分别消化后接种至12孔板,融合达到60%-80%时进行pCAG-T7-CD28-CD137-CD19-CD3-gRNA转染。取2μl重组质粒加入2μl Easyfect,轻轻吹打,摇晃混匀,室温孵育20分钟。用筛选培养基筛选出基因插入的混合克隆细胞株,测序分析检测基因转染效果。
(e)CAR-T细胞体外扩增培养
根据CAR-T细胞计数结果,用OKM-100细胞培养液调整接种密度为(1-2)×106/ml后加入到含有刺激因子的75cm2细胞培养瓶内,然后再加入10%的自体灭活血浆,至37℃CO2细胞培养箱内培养,培养4-5d待细胞铺满瓶底后,将细胞转入225cm2细胞培养瓶中继续培养,培养2-3d后,将225cm2细胞培养瓶中的细胞转至含有1000ml OKM-200细胞培养液的CO2透气培养袋中继续培养,培养6-7d后,收获细胞,计数。
(f)CAR-T细胞回输患者体内,进行抗肿瘤治疗
培养好的CAR-T细胞进行细菌、真菌、支原体、外来病毒及内毒素检测无阳性后,混悬于100ml生理盐水中,用带有滤膜的输液器缓慢静滴越1h,回输前可肌注苯海拉明20-40mg。回输过程检测生命体征及毒副反应。每次回输前抽静脉血做外周血淋巴细胞亚群检测。外周血淋巴细胞表型应用流式细胞仪测定,分别测定CD3+、CD28+、CD19+、CD137+、CD3+CD28+CD19+CD137+、CD3+CD28+、CD19+CD137+、CD3+CD28+CD19+、CD28+CD19+CD137+,细胞在淋巴细胞中所占百分比情况,以获得CAR-T细胞在体内更加详细的数据。同时用MTT法检测培养扩增后CAR-T细胞的杀伤肿瘤细胞的活性。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应该以权利要求的保护范围为准。
Claims (8)
1.一种自体CAR-T细胞制备方法,其特征在于:包括步骤如下:
(a)从肿瘤患者体内获得细胞样本并分离和激活,所述样本为T细胞或T细胞的祖细胞;
(b)构建scFv-CD28-CD137-CD19-CD3;
(c)制作pCAG-T7-CD28-CD137-CD19-CD3-gRNA重组质粒
(d)转染T细胞;
(e)体外培养扩增CAR-T细胞群体;
(f)CAR-T细胞回输患者体内,进行抗肿瘤治疗。
2.根据权利要求1所述的自体CAR-T细胞制备方法,其特征在于:所述步骤(a)的具体操作流程为:1)抗凝血管抽取骨髓瘤患者外周血;2)向管中加入红细胞裂解液和等体积PBS,轻轻吹打成细胞悬液;3)另取两支离心管,加入LTS1077淋巴细胞分层液;用吸管吸取细胞悬液,在距离淋巴细胞分层液上方0.5-3cm处将细胞悬液小心而缓慢的加入,使细胞悬液重叠与淋巴细胞分层液上,2000r/min离心5-50min;4)取出离心管,移液管吸去最上层的血浆,移液枪吸取血装层下的单个核细胞置入离心管中,加入PBS,轻轻吹打均匀后再次离心,1500r/min,10min,去掉上清液,共洗涤2-5次;5)向去掉上清后的离心管内加入含灭活胎牛血清、青链霉素和RPMI-1640的培养基放置于37℃细胞培养箱中扩大培养;6)离心,收取外周血单核细胞,置于液氮罐中保存备用。
3.根据权利要求2所述的自体CAR-T细胞制备方法,其特征在于:所述细胞裂解液和PBS等体积,细胞裂解液和PBS的总体积与抽取的外周血体积比为2:1-1:2。
4.根据权利要求2所述的自体CAR-T细胞制备方法,其特征在于:淋巴细胞分层液和细胞悬液的体积比为2:1-1:2;步骤4)中加入PBS的体积为淋巴细胞分层液的1/2-3倍。
5.根据权利要求2所述的自体CAR-T细胞制备方法,其特征在于:步骤5)中加入的浓度分别是10%灭活胎牛血清、100U/ml青链霉素、100U/mlIL-2的RPMI-1640。
6.根据权利要求1所述的自体CAR-T细胞制备方法,其特征在于:所述步骤(c)为根据CRISPR/Cas9靶点设计原则,应用crispr在线工具设计、合成靶向scFv–CD28-CD137-CD19-CD3全长基因的gRNA和引物,并将其插入到CRISPR/Cas9质粒骨架载体中,制作成pCAG-T7-CD28-CD137-CD19-CD3-gRNA重组质粒。
7.根据权利要求1所述的自体CAR-T细胞制备方法,其特征在于:所述步骤(d)为将步骤(c)制得的质粒CAG-T7-CD28-CD137-CD19-CD3-gRNA与步骤(a)制得的外周血单核细胞混合进行转染。
8.根据权利要求4所述的CAR-T细胞制备方法,其特征在于:所述步骤(e)的详细步骤为:用OKM-100细胞培养液调整接种密度为(1~2)×106/ml后加入到含有刺激因子细胞培养瓶内,然后再加入自体灭活血浆,至细胞培养箱内培养,培养4~5d待细胞铺满瓶底后,将细胞转入大的细胞培养瓶中继续培养,培养2~3d后,将大的细胞培养瓶中的细胞转至含有更大的OKM-200细胞培养液的CO2透气培养袋中继续培养,培养6~7d后,收获细胞。
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